Abstract Although loss of TAR DNA-binding protein 43 kDa (TDP-43) splicing repression is well documented in postmortem tissues of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), whether this abnormality occurs during early-stage disease remains unresolved. Cryptic exon inclusion reflects loss of function of TDP-43, and thus detection of proteins containing cryptic exon-encoded neoepitopes in cerebrospinal fluid (CSF) or blood could reveal the earliest stages of TDP-43 dysregulation in patients. Here we use a newly characterized monoclonal antibody specific to a TDP-43-dependent cryptic epitope (encoded by the cryptic exon found in HDGFL2 ) to show that loss of TDP-43 splicing repression occurs in ALS–FTD, including in presymptomatic C9orf72 mutation carriers. Cryptic hepatoma-derived growth factor-like protein 2 (HDGFL2) accumulates in CSF at significantly higher levels in familial ALS–FTD and sporadic ALS compared with controls and is elevated earlier than neurofilament light and phosphorylated neurofilament heavy chain protein levels in familial disease. Cryptic HDGFL2 can also be detected in blood of individuals with ALS–FTD, including in presymptomatic C9orf72 mutation carriers, and accumulates at levels highly correlated with those in CSF. Our findings indicate that loss of TDP-43 cryptic splicing repression occurs early in disease progression, even presymptomatically, and that detection of the HDGFL2 cryptic neoepitope serves as a potential diagnostic biomarker for ALS, which should facilitate patient recruitment and measurement of target engagement in clinical trials. 

A fluid biomarker reveals loss of TDP-43 splicing repression in presymptomatic ALS-FTD.

Abstract Background Inclusions of TAR DNA-binding protein 43 kDa (TDP-43) has been designated limbic-predominant, age-related TDP-43 encephalopathy (LATE), with or without co-occurrence of Alzheimer’s disease (AD). Approximately, 30–70% AD cases present TDP-43 proteinopathy (AD-TDP), and a greater disease severity compared to AD patients without TDP-43 pathology. However, it remains unclear to what extent TDP-43 dysfunction is involved in AD pathogenesis. Methods To investigate whether TDP-43 dysfunction is a prominent feature in AD-TDP cases, we evaluated whether non-conserved cryptic exons, which serve as a marker of TDP-43 dysfunction in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), accumulate in AD-TDP brains. We assessed a cohort of 192 post-mortem brains from three different brain regions: amygdala, hippocampus, and frontal cortex. Following RNA and protein extraction, qRT-PCR and immunoassays were performed to quantify the accumulation of cryptic RNA targets and phosphorylated TDP-43 pathology, respectively. Results We detected the accumulation of misspliced cryptic or skiptic RNAs of STMN2 , KCNQ2 , UNC13A , CAMK2B , and SYT7 in the amygdala and hippocampus of AD-TDP cases. The topographic distribution of cryptic RNA accumulation mimicked that of phosphorylated TDP-43, regardless of TDP-43 subtype classification. Further, cryptic RNAs efficiently discriminated AD-TDP cases from controls. Conclusions Overall, our results indicate that cryptic RNAs may represent an intriguing new therapeutic and diagnostic target in AD, and that methods aimed at detecting and measuring these species in patient biofluids could be used as a reliable tool to assess TDP-43 pathology in AD. Our work also raises the possibility that TDP-43 dysfunction and related changes in cryptic splicing could represent a common molecular mechanism shared between AD-TDP and FTLD-TDP. 

TDP-43-regulated cryptic RNAs accumulate in Alzheimer’s disease brains

Cryptic exon detection and transcriptomic changes revealed in single-nuclei RNA sequencing of C9ORF72 patients spanning the ALS-FTD spectrum.

Abstract Amyotrophic lateral sclerosis is a complex disorder most of which is ‘sporadic’ of unknown origin but approximately 10% is familial, arising from single mutations in any of more than 30 genes. Thus, there are more than 30 familial ALS subtypes, with different, often unknown, molecular pathologies leading to a complex constellation of clinical phenotypes. We have mouse models for many genetic forms of the disorder, but these do not, on their own, necessarily show us the key pathological pathways at work in human patients. To date, we have no models for the 90% of ALS that is ‘sporadic’. Potential therapies have been developed mainly using a limited set of mouse models, and through lack of alternatives, in the past these have been tested on patients regardless of aetiology. Cancer researchers have undertaken therapy development with similar challenges; they have responded by producing complex mouse models that have transformed understanding of pathological processes, and they have implemented patient stratification in multi-centre trials, leading to the effective translation of basic research findings to the clinic. ALS researchers have successfully adopted this combined approach, and now to increase our understanding of key disease pathologies, and our rate of progress for moving from mouse models to mechanism to ALS therapies we need more, innovative, complex mouse models to address specific questions. 

/pdf/opinion-more-mouse-models-and-more-translation-needed-for-3ikyci12.pdf

Opinion: more mouse models and more translation needed for ALS

Polystyrene nanoparticles trigger aberrant condensation of TDP-43 and amyotrophic lateral sclerosis-like symptoms

Loss of nuclear TDP-43 is a hallmark of neurodegeneration in TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 mislocalization results in cryptic splicing and polyadenylation of pre–messenger RNAs (pre-mRNAs) encoding stathmin-2 (also known as SCG10), a protein that is required for axonal regeneration. We found that TDP-43 binding to a GU-rich region sterically blocked recognition of the cryptic 3′ splice site in STMN2 pre-mRNA. Targeting dCasRx or antisense oligonucleotides (ASOs) suppressed cryptic splicing, which restored axonal regeneration and stathmin-2–dependent lysosome trafficking in TDP-43–deficient human motor neurons. In mice that were gene-edited to contain human STMN2 cryptic splice-polyadenylation sequences, ASO injection into cerebral spinal fluid successfully corrected Stmn2 pre-mRNA misprocessing and restored stathmin-2 expression levels independently of TDP-43 binding. Description Rescue from TDP-43 proteinopathies Loss of the RNA-binding protein TDP-43 from the nuclei of affected neurons is a hallmark of neurodegeneration in TDP-43 proteinopathies, including amyotrophic lateral sclerosis and frontotemporal dementia. The RNA most affected by reduced TDP-43, STMN2, encodes stathmin-2, a protein required for axonal regeneration after injury. Baughn et al. found that TDP-43 sterically blocks recognition of a cryptic splice site in STMN2 pre-mRNA (see the Perspective by O’Brien and Mizielinska). The CRISPR effector dCasRx or antisense oligonucleotides could block STMN2 pre-mRNA cryptic splicing. This approach was able to rescue stathmin-2 levels in TDP-43–deficient human motor neurons and mouse genes edited to contain human STMN2 cryptic splice/polyadenylation sequences. —SMH An antisense oligonucleotide approach shows potential for therapeutic restoration of stathmin-2 in TDP-43 proteinopathies. INTRODUCTION Nuclear clearance and cytoplasmic aggregation of the RNA-binding protein TDP-43 is the hallmark of neurodegenerative diseases called TDP-43 proteinopathies. This includes almost all instances of amyotrophic lateral sclerosis (ALS) and about half of frontotemporal dementia. In ALS, the motor neurons that innervate and trigger contraction of skeletal muscles degenerate, resulting in paralysis. One of the most highly abundant motor neuron mRNAs encodes stathmin-2, a protein necessary for axonal regeneration and maintenance of neuromuscular junctions (NMJs). Loss of functional TDP-43 is accompanied by misprocessing of the STMN2 RNA precursor, which is driven by use of cryptic splicing and polyadenylation sites, and producing a truncated RNA that encodes a nonfunctional stathmin-2 fragment. RATIONALE Recognizing that stathmin-2 is essential for axonal recovery after injury and NMJ maintenance, a central interest in TDP-43 proteinopathies is to determine the mechanism through which TDP-43 enables correct processing of STMN2 mRNAs and to develop methods to restore stathmin-2 synthesis in neurons with TDP-43 dysfunction. RESULTS We found that TDP-43 binding to a 24-base, GU-rich motif within the first intron of the STMN2 pre-mRNA was required to suppress cryptic splicing and polyadenylation. Conversion of this GU-rich binding motif into a 19-base sequence bound by the MS2 bacteriophage coat protein (MCP) ablated TDP-43 binding and produced constitutive misprocessing of STMN2. Correct processing of this modified STMN2 pre-mRNA was restored by binding of MCP, suggesting that TDP-43 normally functions by sterically blocking access to the cryptic sites of RNA-processing factors. Further genome editing revealed that the cryptic 3′ splice acceptor, not the cryptic polyadenylation site, was essential for initiating STMN2 pre-mRNA misprocessing. Rescue of stathmin-2 expression and axonal regeneration after injury in human motor neurons depleted of TDP-43 was achieved with steric binding antisense oligonucleotides (ASOs). Humanization (by insertion of the human STMN2 cryptic exon) sensitized mouse Stmn2 to TDP-43 expression level. Mice alternately humanized with the cryptic exon containing a disrupted TDP-43 binding site produced chronic Stmn2 pre-mRNA misprocessing independent of TDP-43 level. ASOs were identified that when injected into cerebral spinal fluid of mice with constitutive humanized Stmn2 RNA misprocessing, restored stathmin-2 mRNA and protein levels. CONCLUSION We determined that TDP-43 binding in the first intron of the STMN2 pre-mRNA sterically blocked access of RNA processing factors that would otherwise recognize and use a cryptic 3′ splice site. We identified RNA-targeted CRISPR effectors and ASOs that restored STMN2 levels despite reduced TDP-43. ASO injection into cerebral spinal fluid, an approach feasible for human therapy, rescued stathmin-2 protein levels in the central nervous system of mice with chronically misprocessed Stmn2 pre-mRNAs. Steric blocking of cryptic splicing rescues stathmin-2 levels in the central nervous system. (A) TDP-43 sterically blocks recognition of a cryptic splice site in STMN2 pre-RNA, enabling correct processing of intron 1, stathmin-2 protein synthesis, and axonal regeneration after injury. (B) Nuclear clearance of TDP-43 in ALS and other TDP-43 proteinopathies results in cryptic splicing within the first STMN2 intron and loss of stathmin-2 protein. (C) ASOs or targeted RNA-binding proteins can substitute for TDP-43 steric blocking of cryptic splicing within STMN2 pre-mRNAs. (D) Identification of ASOs capable of blocking STMN2 cryptic splicing and restoring stathmin-2 protein levels in the mammalian central nervous system independently of TDP-43 function.

Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies

Genome instability, including chromothripsis, is a hallmark of cancer. Cancer cells frequently contain micronuclei-small, nucleus-like structures formed by chromosome missegregation-that are susceptible to rupture, exposing chromatin to cytoplasmic nucleases. Through an unbiased, imaging-based small interfering RNA screen that targeted all 204 known and putative human nucleases, we identified a previously uncharacterized cytoplasmic endonuclease, NEDD4-binding protein 2 (N4BP2), that enters ruptured micronuclei and initiates DNA damage, leading to chromosome fragmentation. N4BP2 promoted genome rearrangements (including chromothripsis), formation of extrachromosomal DNA (ecDNA) in drug-induced gene amplification, tumorigenesis, and tumor cell proliferation in an induced model of human high-grade glioma. Analysis of more than 10,000 human cancer genomes revealed elevated N4BP2 expression to be predictive of chromothripsis and copy number amplifications, including ecDNA.

Chromothripsis and ecDNA initiated by N4BP2 nuclease fragmentation of cytoplasm-exposed chromosomes.

Abstract Recent studies proposing induced glia-to-neuron conversion raised the potential for generating new neurons to replace those lost due to injury, aging or neurodegenerative diseases. Here, single-cell spatial transcriptomics [ M ultiplexed E rror R obust F luorescence In S itu H ybridization (MERFISH)] is used to construct a spatial cell atlas of the subventricular and dentate gyrus neurogenic niches of young and aged adult murine brain. RNAs that encode the RNA binding protein P olypyrimidine T ract- B inding P rotein (PTBP1) in the aged murine brain are determined to be highest in glia that line previously active neurogenic niches. A glial cell population with ependymal character within an initially quiescent subventricular neurogenic niche in the aged murine brain is identified that upon transient suppression of PTBP1 reenters the cell cycle, replicates DNA, and converts into neurons through a canonical adult neurogenesis pathway. Glia-derived neurons migrate from this niche, with some neurons transiting to the striatum and acquiring a transcriptome characteristic of GABAergic inhibitory neurons. Similar PTBP1 expressing quiescent glia are identified in the corresponding neurogenic niche of aged human brain. Thus, transient reduction of PTBP1 holds potential for inducing the generation of new neurons in quiescent neurogenic niches of the aged nervous system, thereby offering promising therapeutic applications. Bullet point summary 1) Single-cell spatial transcriptomics is used to validate active neurogenesis in the two neurogenic niches of the young adult murine brain, determine that those niches are quiescent in the aging adult brain of mice, and demonstrate the absence of neurogenesis in the aging human brain. 2) The RNA binding protein PTBP1 is determined to be most highly expressed within glia that line the aged murine and human neurogenic niches, with its transient reduction sufficient in mice to activate/re-activate expression of genes characteristic of immature neurons. 3) Suppression of PTBP1 using a single intra-cerebral-ventricular injection of PTBP1–targeting antisense oligonucleotide (ASO) induces generation of new immature neurons in the neurogenic niches of the aged mouse brain via a canonical adult neurogenesis pathway. 4) Single-cell RNA signature tracing is used to identify a) a subclass of ependymal cells in a previously quiescent neurogenic niche of the aged mouse brain that convert into GABAergic inhibitory neurons following transient suppression of PTBP1, and b) the molecular steps in the conversion process including cell cycle re-entry, DNA replication, and transcriptome changes that mimic canonical neurogenesis. 5) A similar class of PTBP1-expressing ependymal cells lining the ventricle of the aging non-human primate and human brains is identified, suggesting the promise of re-activation of neurogenesis as a therapeutic approach in humans. 

Re-activation of neurogenic niches in aging brain

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Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies

Chromothripsis and ecDNA initiated by N4BP2 nuclease fragmentation of cytoplasm-exposed chromosomes.

Re-activation of neurogenic niches in aging brain